Application Note 13 Simultaneously Analyze by HPLC

Benzodiazepines are encountered in a high percentage of In this separation pH is not critical, since similar chromatograms drug screenings done in hospitals. These drugs are used can be obtained at pH 5 or 7. (At pH 3, however, widely to treat anxiety and insomnia. Patients must be coelutes with .) This procedure also eliminates the monitored to evaluate compliance with a therapeutic need for prolonged column equilibration, a characteristic of alternative analyses involving ion pairing reagents (1). regimen and to detect drug abuse. Chlordiazepoxide, , and five active metabolites often prescribed Separating benzodiazepines and their active metabolites is independently as anxiolytic drugs can be separated in only equivalent to monitoring seven separate anxiolytic drugs, and is 12 minutes by HPLC. (ChromFax: 394013) thus unusual. As shown in Figure A, chlordiazepoxide is sequentially demethylated, deaminated, and reduced. Diazepam, on the other hand, may initially be either demethylated or Key Words: hydroxylated. All of the metabolites are eventually converted to benzodiazepines l chlordiazepoxide l diazepam oxazepam. Although other investigators have separated similar l anxiolytic drugs combinations of benzodiazepines by HPLC (2-5) or GC (6), we believe this to be the first simultaneous separation of these seven HPLC analyses for benzodiazepines are preferable to gas chroma- drugs. tography. GC analyses are complicated by the need to simulta- When separating benzodiazepines or other drugs in samples neously quantify the ingested drug and several pharmacological- derived from physiological fluids, we recommend using a guard ly active metabolites (Figure A). Further complicating these column to protect the analytical column. analyses is the thermal instability of some of the compounds, Samples can be cleaned and concentrated by using 1mL particularly chlordiazepoxide and oxazepam. SupelcleanTM LC-18 solid phase extraction tubes. Using the following procedure, all eight benzodiazepines and metabolites Figure A. Chlordiazepoxide, Diazepam, and Pharmacologically Active Metabolites Figure B. Seven Benzodiazepines Analyzed Simultaneously CH HNCH CH3 3 3 O O Column: SUPELCOSIL LC-8, 15cm x 4.6mm, 5µm particle N N C N C C Cat. No.: 58220-U CHOH CH2 CH2 Mobile Phase: :acetonitrile:0.005M KH2PO4 and 0.1M CI C N CI C N CI C N ammonium acetate buffer (pH 6.0 with glacial acetic O acid), 26.5:16.5:57 (v/v/v) Flow Rate: 2mL/min, 2000psig, 30°C Chlordiazepoxide Demoxepam Det.: UV, 245nm, 0.04 AUFS Sample: 10µL containing 40ng each analyte. NH H H H 2 O OO 1 1. Demoxepam 2. (int. std.) N C N C N C N C 3. N-desmethyl Chlordiazepoxide CH CH CHOH 2 CH2 2 3 4. Oxazepam CI C N CI C N CI C NCI CN 5. Chlordiazepoxide O 6. Temazepam O 7. N-desmethyl Diazepam 8. Diazepam N-desmethyl Chlordiazepoxide Diazepam N-desmethyl Diazepam Oxazepam 5 7 2 4 713-1389

6 The HPLC analysis described here includes chlordiazepoxide, 8 diazepam, and five active metabolites prescribed independently as anxiolytic drugs. In Figure B, we separated these benzodiaz- epines and an internal standard (nitrazepam) on a 15cm x 4.6mm SUPELCOSILTM LC-8 column. The separation took only 0 2 4 6810 12 14 713-1390 12 minutes. Min

T394013 ©1994 Sigma-Aldrich Co. can be extracted from serum for the HPLC analysis, with average Figure C. Benzodiazepines Recovered from Serum absolute recoveries ranging from 82% to 106%. by Solid Phase Extraction Condition a 1mL Supelclean LC-18 tube with 2mL of methanol, followed by 2mL of deionized water, then 500µL of 0.001M KOH Column: SUPELCOSIL LC-8, 15cm x 4.6mm, 5µm particle in water. The packing should remain wet during conditioning. Cat. No.: 58220-U Mobile Phase: 0.005M KH2PO4 and 0.1M ammonium acetate Interrupt the flow and add 500µL of deionized water, followed (pH6):methanol:ACN, 57:26.5:16.5 by 100µL of 5% ethanol in water, then 500µL of the serum Flow Rate: 2.0mL/min Temp.: Ambient sample. The order of addition is important to maximize recov- Det.: UV, 245nm

eries. Resume flow and draw the sample mixture through the Initial serum concentrations: tube in a slow dropwise manner, using a VisiprepTM Solid Phase Supelguard LC-8 Column (2cm) 1. Demoxepam (100ng/mL) n 2. Nitrazepam (200ng/mL) Extraction Vacuum Manifold. Wash the packing with 500µL of 3. N-desmethyl Chlordiazepoxide (200ng/mL) 4. Oxazepam (400ng/mL) 0.01M H3PO4. This step should be completed as quickly as 5. Chlordiazepoxide (200ng/mL) possible; a prolonged wash can reduce recoveries. Wash the 6. Temazepam (400ng/mL) 1 2 7. N-desmethyl Diazepam (200ng/mL) packing with 1mL deionized water, then dry for 5-10 minutes 4 8. Diazepam (200ng/mL) l 5 7 under , using a VisidryTM Drying attachment. Elute 3 samples with 400µL of methanol, then evaporate the eluant to 6 dryness under nitrogen. Reconstitute with 200µL of mobile 8 phase prior to analysis. 713-1391

Table 1. Benzodiazepines Recovered from Serum Non-Spiked Extracted Serum by Solid Phase Extraction Absolute Recovery (%) Coefficient 1 2 3 Mean (%) of Variation Demoxepam (100ng/mL) 92 91 90 91 1.1 Nitrazepam (200ng/mL) 107 105 104 105 1.5 N-desmethyl Chlordiazepoxide (200ng/mL) 97 93 95 95 2.1 Oxazepam (400ng/mL) 88 84 84 85 2.7 Chlordiazepoxide (200ng/mL) 98 96 98 97 1.2 0 4812 16 20 713-1392 Min Temazepam (400ng/mL) 83 81 86 83 3.0 N-desmethyl-Diazepam (200ng/mL) 108 104 106 106 1.9 Diazepam (200ng/mL) 88 88 93 90 3.2 References This extraction provides clean samples with good recovery and 1. Sohr, C.J. and A.T. Buechel, J. Anal. Toxicol., 6, 286-289 (1982). 2. Marks, J., The Benzodiazepines: Use, Overuse, Misuse and Abuse, reproducibility that can be analyzed with the HPLC system. Table University Park Press, Baltimore, MD (1978). 1 shows the recoveries at levels that might result from routine 3. Good, T.J. and J. S. Andrews, J. Chromatogr. Sci., 19, 562-566 (1981). dosage administration. Minimum levels quantitated for chlordiaz- 4. Tjaden, U.R., M.T.H.A. Meeles, C.P. Thys, and M. Van Der Kaay, J. epoxide and diazepam were 100ng/mL. Figure C shows a repre- Chromatogr., 181, 227-241 (1980). 5. Vree, T.B., A.M. Baars, Y.A. Hekster, and E. Van Der Kleijn, J. Chromatogr., sentative extracted sample and a serum blank. 224, 519-525 (1981). SUPELCOSIL LC-8 Column, 15cm x 4.6mm 58220-U 6. Loscher, W., Therapeutic Drug Monitoring, 4, 315-318 (1982). SUPELCOSIL LC-8, 25cm x 4.6mm 58297 Acknowledgment: TM 2cm Supelguard LC-8 Columns, Drs. W.E. Scott (Hoffman-LaRoche), P. Russell (Wyeth Labs), and K.D. Roskaz pk. of 2 (5µm packing) 59562 (Sandoz) provided the standards. Supelguard LC-8 Kit 59052 Pelliguard, SUPELCOSIL, Supelclean, Supelguard, Visidry, and Visiprep are 2cm PelliguardTM LC-8 Columns, trademarks of Supelco, Inc. pk. of 4 (40µm packing) 58953 Pelliguard LC-8 Kit 59643 Supelclean LC-18 Solid Phase Extraction Tubes, 1ml, pk. of 100 57002 Contact our Technical Service Department (phone 800-359-3041 or 814-359-3041, FAX 814-359-5468) Visiprep Solid Phase Extraction Vacuum Manifold 57030-U for expert answers to your questions. Visidry Vacuum Manifold Drying Attachment 57100-U n US Pat. Nos. D.289,861; 4,810,471; other patents pending. l US Pat. No. 4,810,471; other patents pending. Note 13

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